The lens is an avascular, syncytial organ that is dependent on intercellular communication for the maintenance of transparency and tissue homeostasis. Three different connexin proteins (Connexin 43 (Cx43), Connexin 46 (Cx46), Connexin 50 (Cx50)) have been identified in lens fibers. The overall objective of this research proposal is to better understand the role of connexins in the lens. The central hypothesis that will be tested is that both connexin gap junctions and hemichannels play an important role in maintaining lens transparency. There are two specific aims: Aim 1. To understand how structural differences in the N-terminal, first transmembrane spanning domain (M1), and first extracellular domain (E1) contribute to differences in the biophysical properties of wild-type and mutant Cx46 and Cx50 gap junctional channels and hemichannels. We will continue to investigate the functional properties of wild-type and mutant lens connexins expressed in heterologous cell systems. The two connexin specific properties that will be studied are: (1) molecular permeability to larger permeants; (2) hemichannel gating. In addition, the functional and cellular effects of several congenital cataract associated connexin mutations that localize to the N- terminal and E1 domains will be tested. Aim 2. To determine the molecular identity of the nonselective leak conductance in fiber cells that is activated by removal of external calcium. Recently, my laboratory has developed a technique for producing a viable, isolated fiber cell preparation from lenses of adult and neonatal mice. We will use this preparation as well as HeLa cells transfected with different lens connexins to address the following questions: What is the molecular identity of the nonselective leak conductance activated by removal of external calcium?; Can this conductance account for the influx of sodium that occurs in fiber cells under physiological conditions?; Does this conductance mediate the pathological calcium influx that occurs in fiber cells in response to cell swelling? The hypothesis to be tested is that connexin hemichannels are responsible for all of these phenomena. We will also use the isolated fiber cell preparation to study the regulation of gap junctional conductance by a variety of factors that have been previously shown to play an important role in regulating intercellular communication in the intact lens such as phosphorylation, oxidative stress and pHi.